Project Summary The digestion and absorption of nutrients from maternal milk during the postnatal period of suckling is fundamentally different from the ?mature? digestion and absorption of ingested food that develops around weaning and takes place henceforth. In adults, proteins from ingested foods are digested by proteases in the lumen of the digestive track, largely in the stomach, and then the resulting amino acids are absorbed by intestinal enterocytes. During suckling, however, the stomach is not yet acidic and proteins pass through it intact. Then, in the intestinal lumen, enterocytes endocytose the whole proteins and degrade them in their lysosomes. For this form of intracellular digestion, enterocytes during the suckling period develop a prominent system of endosomes and lysosomes. We found that two endolysosomal ion channels, mucolipins 3 and 1 (also known as TRPML3 and 1), are highly co-expressed in neonatal enterocytes from birth to weaning. We also found that, in the absence of both mucolipins, neonatal enterocytes suffer pathological endolysosomal vacuolation together with reduced endocytosis, and that the mutant mice suffer diarrhea and reduced growth. All these symptoms disappear by weaning, when pathological enterocytes are replaced by normal-looking, adult-like enterocytes (which do not use endosomes and lysosomes for nutrient absorption). Hence, we have an animal model demonstrating the pathological effects of endolysosomal dysfunction to the neonatal intestine. We will use these and related mice to determine which type of nutrient uptake is compromised upon endolysosomal system failure in neonatal enterocytes. We will also determine with time-inducible and chimeric mutant generation, as well as with viral transfection of cultured enteroids, how many enterocytes, and at what postnatal stages, require endolysosomal functioning to sustain a healthy intestinal function. In order to translate these findings to humans, where the onset of weaning is not clearly defined as it is in rodents and even the existence of suckling-style enterocytes is largely ignored, we will also establish the postnatal period when human enterocytes display a specialized endolysosomal system. We will finally obtain, examine and culture human intestinal biopsies to test the novel hypothesis that endolysosomal abnormalities underlie neonatal intestinal pathologies such as pediatric malabsorption syndromes and necrotizing enterocolitis. These studies may lead to a fundamental change in the understanding of neonatal intestinal pathologies, leading causes of infant mortality worldwide.